Fluorescence is the emission of light by a substance that has absorbed light and in most cases the emitted light has a longer wavelength and lower energy than the absorbed light. Most commonly used fluorescent substances are fluorescent dyes or quantum dots. Limitations with organic fluorescent dyes can be explained as follows: they have absorbance at specific wavelengths, they require multiple excitation wavelengths if multiple dyes are used; their broad emission profile, which causes spectral overlap limiting the production of large number of optical codes including different dyes and lastly, their photo-bleaching, luminescence quenching and low molar extinction coefficient. Quantum dots (QDs), however, with their large absorbance cross-section, narrow symmetric emission band, high molar extinction coefficient, long luminescence, high quantum yield and high resistance to photo-bleaching presents a strong alternative for tagging against fluorescent dyes and colorants.
Fluorescence has many practical applications including fluorescent labeling, tagging, dyes, biological detectors and chemical sensors etc. It is common to use fluorescent substances for variety of fluid products. These fluorescent substances are mixed into the liquid products in the form of fluorescent dyes, quantum dots or colorants and they enable identification of the fluid using optical sensors. This is also a widely accepted methodology in biotechnology, where fluorescent labels are generally used for detection of a protein or other labeled molecule via a fluorescence microscope, flow cytometer or some other fluorescence reading instruments. Such methods can be useful in localization of a target within a cell, flow cytometry (FACS) analysis, western blot assays, and other immune-analytical methods. However the detection of fluorescent markers in these methods requires taking a sample from the fluid and analyzing it with a suitable bulky laboratory apparatus, which is referred to as off-line identification, and this approach is generally inconvenient and time consuming.
Fluorescent substances are also widely used for identification of goods. Tagging of products is highly desirable for manufacturers to solve the problem of identifying, tracking as well as to prevent counterfeiting, product adulteration, unauthorized distribution and sale of products as well as false liability based on product substitution. Fluorescent substances are usually blended in ink and applied to the solid products to create a hologram or a barcode. In some applications, fiber optic probes are used for optically reading the tags. Fiber optical probes introduce advantages of reading from a small volume and improving the sensitivity. However, in such application examples, the reading part includes spectrometer and computers as signal processing resulting in bulky systems.
Bulky readout apparatus that are used in the aforementioned applications are required for sensitive reading of the fluorescent substances in the sub-ppm concentration ranges. However, such bulky systems only allow off-line identification and this is not practical in most of the aforesaid applications. Compact, lightweight and mobile systems which have sensitivity levels better than the bulky systems are highly desirable for online and point of use identification applications in order to give instant results and transfer the results to allocated systems.
In addition to this, in most of the applications, fluorescent substances are blended into fluids which have also their own fluorescence such as ink, petroleum based products etc. A background fluorescence effect is well known for highly absorbent and fluorescent environment/mediums when their emission/absorbance spectrum overlaps with the fluorescent substance emission wavelengths. This leads to difficulties in detection and identification of fluorescent substances present in such mediums.
Fluorescent substances are used previously to tag liquid products and identify those using optical sensors. For instance, U.S. Pat. No. 6,312,958 B1 relates to a method of marking liquids and detecting markers in liquids by exciting fluorescent markers and collecting the emission data from them. In addition, U.S. Pat. No. 5,928,954 describes a method for tagging hydrocarbons and for detecting the presence of tagged hydrocarbons. It is also mentioned in this reference that hydrocarbons have fluorescence and it has to be minimized. In addition, it is mentioned that the excitation results Rayleigh scattering signal creating background interference. However, this invention involves bulky optic setups for reading that is not convenient for online and point of use detection. Also, the reference does not offer any solution for the background signals.
QDs and fluorescent dyes are used for security and identification applications in U.S. Pat. No. 6,692,031 B2, US 20040262400 A1 and U.S. Pat. No. 6,576,155 B1 references. These references blend fluorescent nanocrystals in ink and apply them for tagging solid products. Also, the references use different wavelengths and intensities in order to create a barcode. However, they do not give any information on the background effects or cross talk on multiple markers. Moreover, optical readings in these references comprise bulky and expensive optical setups. Although the US 20040262400 A1 uses a fiber optic sensor for excitation and collection, the apparatus has spectrometer and PC for signal processing that limits the point of use applications.
EP 1441227 A2 reference describes a method, measuring the QDs during the flow. In this reference, fluorescent nanoparticles are placed into beads. During the flow from a channel, beads are excited optically or electrically and their emissions are captured and processed to identify the tag. Effect of QDs with different wavelengths and the effects of the background are not taken into consideration.
Biological applications of fluorescent markers are also discussed in US 20060173362 A1 and WO 01077391 A1 references, in which they are used for identifying cells and for reading beads in multi-assay form respectively. Detection principles in these references are similar but they are mainly for tagging beads for biological applications. Also, they did not mention multiple wavelength fluorescent substance effects on each other, or background effects.
Moreover, sensitivity increase in fiber optical probes is desired in fluorescent substance detection. For instance, US 20080002927 A1 discusses various fiber optic probe assemblies for spectroscopic examinations of biological tissues in-vivo. U.S. Pat. No. 5,878,178 A1, on the other hand, discusses making the tip of the fiber cone in order to increase collection angle. Application of these fiber probes are mainly for biological imaging. US 20120301872 A1 also discusses to improve sensitivity in fluorescent microscopes by placing a retro reflector below the sample carrier. However these techniques will also improve the noise from the background and it is not discussed in these references.
In WO2008/019448 time gating is used for flow cytometry to capture only long lived fluorescence emission after auto-fluorescence has decayed away. However this is mainly used for flow cytometry and the system is bulky.